Glycolic acid (HOCH2COOH) is the simplest member of the alpha-hydroxy acid family of carboxylic acids. Glycolic acid has dual functionality with both alcohol and moderately strong acid functional groups on a very small molecule. This results in unique chemical attributes as well as typical acid and alcohol chemistry.
Glycolic acid uses both the hydroxyl and carboxylic acid groups to form five-member ring complexes (chelates) with polyvalent metals. This metal ion complexing ability is useful in dissolution of hard water scale and prevention of deposition, especially in acid cleaning applications where good rinsibility is a key factor. Its properties make it ideal for a broad spectrum of consumer and industrial applications, including use in water well rehabilitation, the leather industry, the oil and gas industry, the laundry and textile industry, and as a component in personal care products. Glycolic acid undergoes reactions with organic alcohols and acids to form esters. Low molecular weight alkyl glycolic esters have unusual solvency properties and may be used as a substitute for n- and iso-propanol, ethylenediamine, phenol, m-cresol, 2-ethoxyethyl acetate, and ethyl and methyl lactate. Higher molecular weight alkyl esters can be used in personal care product formulations.
Glycolic acid can also be used to produce a variety of polymeric materials, including thermoplastic resins comprising polyglycolic acid. Resins comprising polyglycolic acid have excellent gas barrier properties, and such thermoplastic resins comprising polyglycolic acid may be used to make packaging materials having the same properties (e.g., beverage containers, etc.). The polyester polymers gradually hydrolyze in aqueous environments at controllable rates. This property makes them useful in biomedical applications such as dissolvable sutures and in applications where a controlled release of acid is needed to reduce pH. Currently more than 15,000 tons of glycolic acid are consumed annually in the United states.
The biological production of glycolic acid from an inexpensive carbon substrate such as glucose or other sugars, presented in FIG. 1, is disclosed in WO 2007/140816 and WO 2007/141316 respectively which content is incorporated herein by reference. The microorganism described in these applications is genetically engineered at different levels:                to enhance the flux in the glyoxylate pathway,        to increase the conversion of glyoxylate into glycolate and,        to reduce the metabolism of glycolate and it intermediate, the glyoxylate.        
The modifications have a direct impact on the terminal reactions of the glycolate synthesis pathway or on the closest intermediates of the glycolate. They all have the same objective, to direct the carbon flux at the production of glycolic acid and to prevent the catabolism of it.
The biological production of glycolic acid requires the formation of glyoxylate as an intermediate which is reduced to glycolate by a NADPH dependent oxidoreductase encoded by the gene ycdW (Nunez et al, (2001) Biochemistry, 354, 707-715). Glyoxylate is an intermediate of the glyoxylate cycle, a shunt of the TCA cycle (Tricarboxylic acid cycle and glyoxylate bypass, reviewed in Neidhardt, F. C. (Ed. in Chief), R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low, B. Magasanik, W. S. Reznikoff, M. Riley, M. Schaechter, and H. E. Umbarger (eds). 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology). Before getting into the TCA cycle, the carbon flux goes through glycolysis where several reactions take place and could be optimized to improve the production of the desired compound.
Glycolysis is a sequence of ten reactions involving ten intermediate compounds that converts glucose into pyruvate. The intermediates provide entry points to glycolysis and may also be directly or indirectly useful. For example, the intermediate dihydroxyacetone phosphate (DHAP) is a source of lactate via the protein MgsA. It is the same for the pyruvate molecule converted to lactate by the lactate dehydrogenase, LdhA. Both enzymes consume molecules of the glycolytic pathway meaning a part of the carbon flux to produce lactate, an undesired by-product in that case. The gene ldhA is attenuated in processes for production of succinate, such as in a rumen bacterial strain (WO2008/013405A1) or in E.coli for succinate production with a high yield. On the contrary, ldhA is overexpressed in processes aimed for the synthesis of lactate U.S. Pat. No. 7,700,332. The equivalent is found for mgsA, which is deleted among other genetic modifications for the production of the 1,3-propanediol (WO2004/033646A2), but overexpressed for the synthesis of the 1,2-propanediol (WO02008/116852A1). By attenuating both activities the production of glycolic acid should be improved and as the same time the synthesis of lactate should be reduced.
In the same manner every genetic modification that rises glycolytic and TCA fluxes, glucose import or catalytic activity of glycolytic and TCA enzymes would improve the production of glycolate. The attenuation of ArcA activity is one of such mutation. Indeed, the protein was shown as to be involved in repression of genes encoding the enzymes mentioned above.
The understanding of the global regulation by the Arc system started with the paper by Iuchi and Lin in 1988 (Iuchi and Lin, 1988, PNAS; 85: 1888-1892) describing the identification of the arcA gene and the effect of its mutation on the aerobic metabolism in E. coli. The two-component signal transduction system ArcAB (aerobic respiration control) modulates, at the transcriptional level, the expression of between 100 and 150 operons involved in energy metabolism, transport, survival, catabolism and in the redox state of the cell (Liu and DeWulf 2004, J Biol Chem, 279:12588-12597; Lynch and Lin, 1996 Responses to molecular oxygen; in Neidhardt F C, Curtiss III R J, Ingraham L, Lin ECC, Low K B, Magasanik B W, Zeznikoff S, Riley M, Schaechter M, Umbarger H E (eds) Escherichia coli and Salmonella: Cellular and Molecular Biology, ed 2. Washington, American Society for Microbiology, 1996, vol 1, pp 1526-1538). The main function of the ArcAB signal transduction pair is to regulate the transition from aerobic to anaerobic pathways in E. coli. Further understanding of the correlation between the aerobiosis level and the control exerted by global regulation was obtained from the work of Shalel-Levanon and colleagues (Biotechnol. Bioeng. 2005a; 89:556-564 and Biotechnol. Bioeng. 2005b; 92:147-159). These studies had shown the repression of the TCA genes by ArcA. These results were confirmed by complete physiological studies on the effect of ArcA on glucose catabolism in E. coli at different conditions of oxygen availability. Several changes have been observed in a ΔarcA mutant and under microaerobiosis; such as increased respiration, an altered electron flux distribution over the cytochrome o- and d-terminal oxidases, and a modification in the intracellular redox state (Aleexeva et al., 2003, J. Bacteriol. 185:204-209). The work of Perrenoud and Sauer in 2005 provided new insights onto ArcAB regulation, demonstrating that the control of aerobic and fully anaerobic TCA cycle fluxes was exerted by ArcA independently of its cognate sensor kinase, ArcB (J. Bacteriol. 2005, 187:3171-3179).
All those make ArcA a global regulator in E. coli. Its deletion is described in several patents claiming aerobiosis or anaerobiosis production of a desired molecule. For instance, ΔarcA improves the production of succinate and 1,2-propanediol in aerobiosis as in anaerobiosis (US 2006/0073577A1; WO2006/020663 and WO2008/116848). A decrease of ArcA activity among other genetic modifications was also disclosed in patents for the production of L-amino acid such as L-lysine and L-glutamic acid by Ajinomoto (EP 1 382686A1) and for the production of 1,3-propanediol (WO2004/033646) by DuPont de Nemours and Co.
Three proteins named GlcA, LIdP and YjcG were characterized in the litterature as importers of glycolate and lactate (Nunez, F. et al., 2001 Microbiology, 147, 1069-1077; Nunez, F. et al., 2002 Biochem. And Biophysical research communications 290, 824-829; Gimenez, R. et al., 2003 J. of Bacteriol. 185, 21, 6448-6455). According to the publications mentioned above, GlcA seems to be more specific of glycolate and Lldp has more affinity for the lactate molecule. A strain wherein the three glycolate permeases are deleted is totally unable to import exogenous glycolate (Gimenez, R. et al., 2003 J. of Bacteriol. 185, 21, 6448-6455). The attenuation of the glycolate import of the strain producing glycolate would improve the ability of the cell to resist to high concentrations of glycolate and so, to improve the titer of production.
The problem to be solved by the present invention is the improvement of the biological production of glycolic acid from an inexpensive carbon substrate such as glucose or other sugars. Additional genetic modifications and in particular combination of them, are here described to get much better yield and titer of production of glycolic acid by fermentation.